Nucleotide sequence determination is an important step in the analysis of a short strand of an unknown oligonucleotide or oligonucleotide analog and to confirm the specific sequence of oligonucleotides used as antisense drugs. At the present, most sequencing protocols use the chemical degradation approach of Maxam et al. (Proc. Natl. Acad. Sci. (USA) (1977) 74:560) or the chain-termination method of Sanger et al. (Proc. Natl. Acad. Sci. (USA) (1977) 74:5463). In these methods, four separate reactions are performed to yield fragments differing in length by only a single nucleotide which terminate at adenosine, cytosine, guanosine, or thymidine residues.
These sequencing products are generally resolved by electrophoresis on denaturing polyacrylamide gels (PAGE). High performance capillary electrophoresis (HPCE) has also been used to separate oligonucleotide sequencing products (Cohen et al. (1988) J. Chromatogr. 458:323; Cohen et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:9660; Guttman et al. (1990) Anal. Chem. 62:137; Cohen et al. (1990) J. Chromatogr. 516:49; Cohen et al., Anal. Chem. (in press)), and can be readily coupled to mass spectrometry (Smith et al. (1988) Anal. Chem. 60:1948; Lee et al. (1988) J. Chromatogr. 457:313). However, traditionally, the method of product visualization has been autoradiography wherein .sup.32 p or .sup.35 S is incorporated into the oligonucleotide strand.
Recently, sequencing with laser-induced fluorescence (LIF) as a detection mode has been used in a variety of Sanger et al.-related protocols. In the basic method, four unique fluorescent tags are attached either to the primer (Smith et al. (1986) Nature 321:674) or to each of the terminating dideoxynucleotides (Prober et al. (1987) Science 238:336). In other Sanger et al.-related protocols, single-dye-based coding of bases with four different peak heights has been used (Tabor et al. (1990) J. Biol. Chem. 265:8322-8326; Ausorge et al. (1990) Nucleic Acids Res. 18:3419-3420; Pentoney et al. (1992) Electrophoresis 13:461-474); Huang et al. (1992) Anal. Chem. 64:2149-2154), as well as single-dye-based coding of bases by peak height ratios plus one base coded by a gap (Ausorge et al. (1990) Nucleic Acids Res. 18:3419-3420; Pentoney et al. (1992) Electrophoresis 13:461-474), and two-dye-binary coding of three bases with one base coded by a gap or two optical channels (Carson et al., Anal. Chem. (in press)).
Unfortunately, many oligonucleotides and oligonucleotide analogs such as those useful for the antisense chemotherapeutic approach are too short to be sequenced by conventional sequencing methodologies. For example, if one uses the Sanger et al. approach to sequence a short (e.g., 15 to 17 bases in length), single-stranded DNA, the base sequence at its 3' end is lost. The loss of information is primer size-dependent and normally 15 to 17 bases, i.e, sequence information will be provided right after the primer only.
Nevertheless, correct sequences are required for efficacy, and quality control procedures are needed to ensure that synthetic oligonucleotides have the desired nucleotide sequences. At present, the sequences of such oligonucleotides are often assumed to be correct based on the step-by-step synthesis itself since there is no convenient method available for their sequence analysis.
Enzymatic sequencing of short DNA analogs has been documented (Nordhoft et al. (1992) Rapid Comm. Mass. Spectrom. 6:771; Wu et al. (1993 ) Rapid Comm. Mass. Spectrom. 7:142; and Rile et al. (1993) Rapid Comm. Mass. Spectrom. 7:195). This method uses exonucleases with phosphodiester-linked DNA as a substrate and MALDI-MS for detection. The current protocol is relatively slow, as aliquots are taken every 15 minutes and directly analyzed by MALDI-MS (Tabor et al. (1990) J. Biol. Chem. 265:8322-8326). In addition, when DNA analogs are sequenced under these conditions, exonuclease digestion is very problematic and sometimes impossible.
An added level of complexity is the presence of modifications in oligonucleotides including non-phosphodiester linkages such as phosphorothioates or alkylphosphonates. Previous method of analyzing such oligonucleotide analogs have been laborious for commercial application. For example, Agrawal et al. (J. Chromatogr. (1990) 509:396-399) discloses analysis of oligonucleotide phosphorothioates involving conversion of phosphorothioate linkages to phosphodiesters followed by digestion with snake venom phosphodiesterase, phosphatase treatment, and analysis of base composition on reversed phase HPLC.
Thus, there remains a need for more simple and reliable methods of determining the sequence of short oligonucleotides and oligonucleotide analogs from their very first to their very last base.